The present invention relates to the use of Phyllanthus for preventing or treating connective tissue proliferations, for maintaining the level of reduced glutathione, for inhibiting the lipopolysaccharide (LPS)-induced nitric oxide synthase (NOS) and for inhibiting the expression of the cyclooxygenase (COX-2) protein.
Phyllanthus embraces a widespread group of plants native to Central and South India, Taiwan, and areas of Central and South America. The term Phyllanthus means for the purpose of this invention all representatives of the botanical family of Phyllanthus, such as Phyllanthus niruri or, in particular, Phyllanthus amarus etc. The treatment of a large number of disorders with Phyllanthus is known in Indian folk medicine. Thus, for example, the author of xe2x80x9cDoctor K. M. Nadkarni""s Indian Materia Medica (3rd edition; revised and enlarged by A. K. Nadkarni)xe2x80x9d reckons in volume I that the plant is known to be deobstruent, diuretic, astringent and cooling. Likewise, compositions with Phyllanthus are described for treating jaundice, dropsy, gonorrhoea, menorrhagia and other impairments of a similar type relating to the urogenital tract. Also known are the use of the sap from the trunk mixed with oil as ophthalmologicals or administrations for ulcers, wound sites and swellings etc., as well as the leaves for treating pruritus or other skin impairments.
There is also known to be a large number of active substances which can be isolated from Phyllanthus, phyllanthin, hypophyllanthin, triacontanol, triacontanal, repandusinic acid A (see, for example, JP 03206044 A; AIDS-Res-Hum-Retroviruses (11/1992), vol. 8 (11), HIV-1 reverse transcriptase . . . ), phyllanthostatin-1, phyllanthoside, phyllanthocin, phyllanthocin acid (see, for example, EP 173 4480; U.S. Pat. No. 4,388,457), phyllamycin A, B and C, retrojusticidin B, justicidin A and B (see, for example, AIDS-Weekly, 25.9.95, AIDS Therapies Extracts . . . ), linoleic acid, linolenic acid and ricinoleic acid (see, for example, Journal of the American Oil Chemists Society edition 81.06.00, ricinoleic acid in Phyllanthus niruri . . . ), phyllamyricin D, E and F, phyllamyricoside A, B and C (see, for example, J. Nat. Prod. (11/1996), vol. 59 (11), Six lignans from . . . ), putranjivain A (see, for example, Chem. Pharm. Bull. (Tokyo), (04/1995), vol. 43 (4), Inhibitory effects of Egyptian . . . ), ursulic acid and niruriside (see, for example, J. Nat. Prod. 02/96, vol. 59 (2), Niruriside, a new HIV . . . , Rec. Trav. Chim. (06/1996), Synthesis of . . . ).
Therapeutic effects and administrations disclosed to date are an age-retarding effect (see, for example, JP 08176004), prevention and therapy of immunodeficiencies such as AIDS, influenza, colds, tuberculosis, hepatitis, cirrhoses (see, for example, U.S. Pat. No. 5,529,778; AIDS-Weekly-Plus of 05.08.96, Antiviral (Drug Development); Inhibition of HIV . . . ), antineo-plastic effect (see, for example, U.S. Pat. No. 4,388,457), therapy of HIV, HBV and/or HCV infections, especially topical treatment of Kaposi""s sarcoma (see, for example, EP 1734480; U.S. Pat. No. 5,466,455), effect as protease inhibitor, elastase inhibitor and as bleach (see, for example, JP 09087136), analgesic and antiinflammatory effect, effect as tyrosinase inhibitor (see, for example, JP 08012566) and a use as disinfectant in combination with extracts from other plants. Moreover, uses in cosmetic preparations are also known. It is evident from this large number of different areas of administration and isolated active substances that Phyllanthus is a thoroughly well-known group of medicinal plants employed for a large number of indications and complaints.
One pathological phenomenon which appears to be responsible for a large number of other complaints is so-called oxidative stress. By this is meant the stress on the living cell through accumulation of toxic oxidized compounds, such as lipid hydroperoxides, hydrogen peroxide, singlet oxygen and hydroxyl/superoxide anions. It is moreover possible for the stress to arise through free radicals which are produced locally or supplied from outside, especially so-called reactive oxygen species (ROS) or peroxonitrite free radicals etc. The oxidative stress can also be induced, for example, by exposure to radiation, xenobiotics, heavy metal ions or ischaemia/reperfusion (temporary interruption of the blood supply to an organ). In the latter case there is copious formation of superoxide anions by xanthine oxidase, one of the oxidases of about 300 kD and belonging to the flavoproteins, which catalyses the breakdown of purines, because its natural electron acceptor is oxygen. Under physiological conditions, these superoxide anions are deactivated by superoxide dismutase, but on reperfusion it has been demonstrated that this involves production of large amounts of oxygen free radicals.
Oxidative stress plays an important part in the development of a number of acute and, in particular, chronic disorders, for example inflammations of various types, microangiopathies, fibrosis, rheumatoid arthritis and other rheumatic disorders, arteriosclerosis (LDL oxidation), tumour development and progression, possibly Alzheimer""s disease, but also drug-induced acute damage such as paracetamol damage to the liver.
The liver moreover plays, as central dynamic organ of the body, an important part in a large number of the physiological and microphysiological processes mentioned, the metabolic activities of the liver (intermediary metabolism) being of crucial importance on the one hand for supplying other organs but also, on the other hand, for the chemical conversion (biotransformation) of pharmaceutically active substances (see Pschyrembel, Klinisches Wxc3x6rterbuch, de Gruyter Verlag, 1986, pp. 935-937). The phenomenon of oxidative stress which has already been described is likewise for the most part combated by the body in the liver. This contains a reservoir of diverse reduced compounds of antioxidants, for example L-ascorbic acid, carotenoids, dihydrolipoic acid, uric acid, glutathione or xcex1-tocopherol, and prevents the occurrence of reactive free radicals by means of various enzyme activities (for example superoxide dismutase, peroxidases such as glutathione peroxidase, catalase, etc.).
Oxidative stress also has in particular adverse effects on a large number of functions of liver tissue. Liver tissue frequently responds to this with connective tissue proliferation, which favours further progression of permanent liver damage, such as, for example, the development of a liver tumour. In this connection, bile acids in particular are involved in the pathogenesis of the hepatotoxic effect.
In all the above-mentioned disorders, the balance between oxidative stress and the defence systems of the cells and organs have crucial importance. It is therefore of crucial prophylactic and therapeutic importance on the one hand to protect the healthy liver from oxidative stress and, on the other hand, to strengthen the diseased liver so that it is able permanently to overcome pre-existing oxidative stress.
Compounds with a hepatoprotective activity have in some cases considerable disadvantages because they cannot be used if the liver is already diseased, because the toxicity is too high.
The present invention is therefore based on the object of providing substances which act on the liver and have both a prophylactic and a therapeutic effect.
The present invention relates to the use of Phyllanthus for preventing or treating connective tissue proliferations, in particular fibrotic changes for example of the liver, of the lung, of the kidney, of the pancreas, of the intestine, of endocrine organs, of the spleen, of the male or female urogenital tract, of the joints, for example as a consequence of chronic inflammatory processes such as, for example, rheumatoid arthritis or chronic cardiomyopathies, and of cirrhoses, an advanced stage of fibroses.
The use according to the present invention is therefore particularly preferable for fibroses and cirrhoses, preferably fibrosis of the liver and cirrhosis of the liver. In this connection in particular chronic inflammatory states lead to tissue atrophy and pronounced scar formation with progressive loss of function of the organs. An inhibition or prevention of the connective tissue proliferation therefore leads to less pronounced scar formation and retention of the ability of the organs to function.
The corresponding activity of Phyllanthus in preventing or improving in particular fibrosis of the liver presumably derives from an antioxidative effect, fibroses of the liver frequently being caused by viral infections. Thus, for example, all the known hepatitis viruses (hepatitis A, B, C, D, E and probably also G) have a pronounced tropism for liver cells. It is to be assumed that even the antiviral medicines currently used in medicine lead not to elimination of viruses but only to suppression of virus replication (virus suppression). Even if the virus disappears from the peripheral blood (below the detection limit), the virus is often still detectable in liver tissue. Thus, Phyllanthus can exert an advantageous effect on liver regeneration through its prophylactic and therapeutic effect. This contributes to reducing chronic inflammatory processes, with a reduction in the developing connective tissue proliferation in the liver. No medicines which can intervene in such an early step of degenerative development have yet been disclosed.
The present invention further relates to the use of Phyllanthus for maintaining the level of reduced glutathione. It has surprisingly been found that Phyllanthus extracts have potent activity in the maintenance of the level of reduced glutathione, which occurs in particular in the liver. In these experiments it was found that, in the functional cells of the liver (hepatocytes) showing increased lipid peroxidation due to t-butyl hydroperoxide, an extract of Phyllanthus not only suppresses further lipid peroxidation but even almost completely abolishes endogenous lipid peroxidation. In comparative experiments with untreated hepatocytes, a clear increase in the reductive capacity was found, which suggests improved maintenance of the intracellular level of reduced glutathione.
In preferred embodiments, Phyllanthus is used to reduce the expression of smooth muscle alpha-actin (SMA) mRNA and SMA protein. In a fibrotic liver, which has an increased rate of cell division, there is accumulation of extracellular matrix. The increased amounts of extracellular matrix are regarded as crucial for further progression of fibrosis of the liver as far as cirrhosis of the liver. The accumulation of extracellular matrix derives from activation of specific liver cells, the hepatic stellate cells (HSC), which in activated form are referred to as activated HSC. Compared with non-activated HSC, activated HSC produce larger amounts of smooth muscle alpha-actin (SMA) mRNA and protein, which is why the activation of HSC can be measured from the expression of SMA. The activation of HSC can also be judged from the expression and intracellular distribution of glial fibrillary acidic protein (GFAP). It has now been surprisingly found in a series of experiments that HSC lead, after treatment with Phyllanthus extracts, to distinctly smaller amounts of extractable SMA mRNA and SMA protein. Furthermore, by using immunofluorescence to detect the distribution of SMA and GFAP it could be demonstrated that extracts of Phyllanthus stabilise the normal phenotype of HSC and prevent the increase in cell size associated with their activated state. In addition, HSC treated with Phyllanthus extracts showed a distinct inhibition of cell growth, which underlines the activity of Phyllanthus extracts in these experiments. This means that there is a possibility of converting activated HSC as occur in fibrotic liver back into non-activated HSC, thus favouring a regression of fibrosis of the liver.
The present invention further relates to the use of Phyllanthus for inhibiting lipopolysaccharide (LPS)-induced nitric oxide synthase (NOS), particularly preferably inhibiting the induced nitric oxide synthase (iNOS), and the use of Phyllanthus for inhibiting the expression of cyclooxygenase (COX-2) protein.
LPS is a collective term for conjugates composed of lipid and polysaccharide portions. The LPS occurring in the outer membrane of the cell wall of Gram-negative bacteria are composed in principle of three components, namely lipid A, the core oligosaccharide and the O-specific side chains. Lipid A anchors the LPS in the bacterial cell wall and is also responsible for the immunoactivating effect of bacterial cell wall constituents.
The expression of iNOS and COX-2 can be induced in liver cells with the aid of LPS, which is why the stimulated cells can be used as model systems for fibrotic liver cells in which the expression of these two proteins is likewise raised. Both iNOS and COX-2 is known as a potent mediator of inflammatory processes like those occurring with degenerative changes of the liver. It has now surprisingly been found in comparative experiments that liver cells stimulated with LPS and then treated with Phyllanthus extracts showed a marked reduction in the rates of expression of the iNOS protein and the COX-2 protein.
This invention further comprises the use in one of the above-mentioned ways, using a fraction isolated from Phyllanthus. An isolated fraction means in this sense a subsidiary amount of Phyllanthus substances which has been removed, for example, by chromatographic means, distillation, precipitation, extraction, filtration or in other ways from Phyllanthus. It means in particular extracts and the fractions removed therefrom by chromatography, distillation, precipitation or extraction.
Another use according to the invention comprises uses in accordance with one of the above-mentioned examples, in which one or more chemical substances, in particular active substances, isolated from Phyllanthus are used. By these are meant in particular also single substances isolated from Phyllanthus extracts or other extracts, so-called natural substance isolates, as are also known, for example, from the prior art. The use of these isolated active substances has the advantage that it is generally necessary to use considerably smaller amounts of substance and, moreover, more specific effects are often achieved than with whole extracts or tablets.
In preferred uses according to one of the use forms according to the invention, Phyllanthus is selected from individual members of the Phyllanthus family, from the group of Phyllanthus amarus, Phyllanthus niruri, Phyllanthus emblica, Phyllanthus urinaria, Phyllanthus myrtifolius Moon, Phyllanthus maderas pratensis and/or Phyllanthus ussuriensis. 
In the uses according to the invention it is possible to use leaves, bark, flowers, seeds, fruits, stalks, branches, trunk, root and/or wood of Phyllanthus, preferably the herb, that is to say all above-ground parts of the plant. It is moreover possible for Phyllanthus to be used in comminuted form and/or in unmodified form, that is to say as whole leaf, as granules, powder, precipitate, extract, dried extract and/or exudate, with extracts or dried extracts being preferred.
The preparation of Phyllanthus for the use according to the invention comprises the preparation of Phyllanthus powder or granules from one of the above-mentioned plant parts, extraction from plants, comminuted plant parts, powders, and residues which have already been treated previously with other solvents, with hexane, water, methanol and/or other alcohols. This also includes filtration and vacuum evaporation in order to obtain a dried extract. Another method comprises multiphase extraction with aqueous and/or alcoholic and/or polar solvents. Also usual is filtration, for example through cellulose filters, precipitation, preferably using ethanol, or separation by ultracentrifugation, and maceration. It is moreover always possible to operate at elevated or reduced temperatures.
It is particularly preferred here to use Phyllanthus in the form of an aqueous, lipophil or alcoholic extract, the alcoholic extract being carried out preferably with short-chain (C1 to C4) primary alcohols or mixtures thereof, especially methanol or ethanol, the lipophil one with C5-C10, branched or unbranched, chain hydrocarbons, or mixtures thereof, especially with n-hexane.
Also suitable as extractants are ethyl acetate or appropriate organic solvent/water mixtures, preferably methanol/water mixtures or ethanol/water mixtures. A suitable extraction process is disclosed, for example, in U.S. Pat. No. 4,673,575 or U.S. Pat. No. 4,937,074.
In uses according to the invention, Phyllanthus is preferably employed in the form of one or more medicinal products (see Rxc3x6mp, Lexikon Chemie, Version 1.4), such as an infusion solution, injection solution, tablet, granules, ointment, medicinal pack, enemas or in the form of one or more foodstuffs/food supplements. These embrace the usual medical and therapeutic applications and, in particular, also those as food supplements, it being possible to use Phyllanthus in these cases for prophylaxis and as functional antioxidant which is a natural and non-hazardous addition to foodstuffs and, moreover, shows the therapeutic and functional preventive effects mentioned.
The use of Phyllanthus in the uses according to the invention can take place orally, topically and/or parenterally.
In a preferred embodiment, one or more other active substances and/or suitable additives and/or auxiliaries are used in addition to Phyllanthus. The term active substance means for the purpose of this invention therapeutically active substances such as, for example, vitamin C or tocopherols, especially xcex1-tocopherol, which are known as antioxidants or as active substances against oxidative stress, and, for example, anti-inflammatory substances. These also embrace therefore so-called combination products with Phyllanthus. It should also be noted in this connection in particular that the uses according to the invention are by no means confined to only one form, fraction or isolated active substance from Phyllanthus in each case, and it is also possible to use different forms and/or fractions and/or isolated active substance from Phyllanthus for a use.
Auxiliaries and additives mean for the purpose of this invention substances which are known to be added for therapeutic applications or as application for food supplements in order to permit or facilitate a corresponding use, for example adjuvants, disintegrants and lubricants, bulking agents, buffers, preservatives, stabilizers etc.
Fractions of Phyllanthus-derived material suitable for use in the methods of this invention may be obtained by dividing or extracting or fractionating Phyllanthus, including without limit leaves, bark, flowers, seeds, fruits, stalks, branches, trunk, root and/or wood, according to the fractionation procedures described above and in the Examples below. Individual procedures may be applied singly or in combination. Variations on these procedures will be apparent to the skilled artisan, in view of the guidance provided herein. Fractions of Phyllanthus may be formulated and adminstered as described herein for Phyllanthus material, including additives and/or auxiliaries. Preferably, fractions of Phyllanthus used in the methods of this invention will be biologically active as determined in one or more of the experimental procedures described herein for identifying and/or quantifying the biological effects of Phyllanthus, especially procedures described in the Examples. Selection of fractions which exhibit such biological activities is within the skill of the ordinary artisan in view of the guidance provided herein.